Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease (AD) and related dementias. NFTs are of growing biomedical interest as the temporal and spatial pattern of NFT appearance in human brain correlates well with AD progression. The tangle is composed of microtubule associated protein tau, which can be hyper-phosphorylated and aggregated. Indeed, tau protein purified from the brains of AD patients is hyper-phosphorylated, which has led to investigations of the role of tau phosphorylation in mediating neurodegeneration. Alzheimer's pathogenic mechanisms still remain elusive. Recently, an intriguing concept of prion-like spreading of tau has emerged, which has the potential to transform AD research. A prion-like mechanism involving the transfer of hyper-phosphorylated tau between synaptically connected neurons underlies the seeding and spread of tau pathology. New insights into the molecular mechanisms of tau propagation will uncover potential therapeutic targets for slowing or even halting AD progression. Hyper-phosphorylation of tau is also known to be involved in tau release, causing its cell-to-cell propagation. However, there is still a significant gap in understanding how phosphorylation regulates tau release. We hypothesize that individual phosphorylation sites will differentially affect tau release and that studying this phenomena will uncover a currently unexplored network of endogenous protein kinases that act to regulate tau release by phosphorylating specific residues on tau. In particular, it is not known what phosphorylation sites of tau (pTau) are crucial for its release and kinases have yet to be identified for their role in tau release - a recognized seminal step in the prion-like spreading of tau protein. In Aim 1, we will study the role of specific phosphorylation sites of tau on its release. pTau sites to be examined will be primarily in proline-rich domain (PRD) and C-terminal region of tau as ~75% of disease associated pTau sites are found in these regions. In Aim 2, we will examine 5 kinases for their role in tau release as they are known to phosphorylate amino acids in PRD and C-terminal regions. Aim 3 will test if activity-driven endogenous hTau release is modulated by phosphorylation of tau at specific amino acids using a human neural cell line (ReNCell) endogenously expressing hTau. Our approach with Drosophila (Aims 1 & 2) and human neuronal culture models (Aim 3) will also provide excellent opportunities for training and mentoring both undergraduate and graduate students.